Introduction
Chemo-therapeutants of non-phytotoxic properties, biodegradable and systemic application are mostly found from plant sources (Chamanlal & Verma, 2006). Such knowledge of these constituents is essential in searching for novel therapeutic agents and the authentification of traditional medicinal claims (Sriram, Vaidyanathan, Venkatesh, & Nayagam, 2011). The increasing prevalence of resistance to standard antibiotic therapy among common pathogens is quite challenging, which is rapidly becoming a major health problem throughout the world (Khan et al., 2006). Infections caused by S. aureus, particularly multidrug-resistant strains and coagulase negative Staphylococcus, Enterococcus, and Pneumococci, were previously challenging to treat (Gale, Johnson, Kerridge, & Wayman, 1980).
S. aureus, a leading cause of skin and soft tissue infections. S. aureus causes various illnesses, including boils, bacterial meningitis, and wound infections, and is frequently involved in nosocomial infections. It is known for its multi-drug resistance, particularly the presence of the mecA gene, which allows it to develop resistance to most -lactam medicines, including methicillin, and is hence known as methicillin-resistant S. aureus (MRSA) (Hiramatsu et al., 2014). E. coli is a typical flora of the human digestive system, but it may become pathogenic when ingested orally, causing diseases ranging from urinary tract infections to gastrointestinal tract infections, bacteremia, and newborn meningitis. K. pneumonia is a gram-negative bacilli, a facultative anaerobe that ferments lactose and is widely found in the colon. It has also been linked to skin and soft-tissue infections.P. aeruginosa is implicated in nosocomial infections, respiratory tract infection (RTI), UTI, GIT, dermatitis, soft tissue infections and infection in patients with cancer, HIV-AIDS and severe burn. Responses to treatment are compromised by inherent multi-drug resistance acquired by the bacteria. It is resistant to β-lactam and aminoglycoside and negatively induces the expression of outer membrane proteins, up-regulation of efflux pumps and can cause mutation in topoisomerases (Matar, 2018). S. typhi is also a gram-negative bacilli, a facultative anaerobe that infects birds, animals and humans. It is a causative agent of typhoid fever. Salmonella enterica serotype Typhi species is very diverse. The disease burden caused by S. typhi was estimated by WHO in 2000 to be approximately 22 million illnesses and 220,000 deaths worldwide (Crump, Luby, & Mintz, 2004).
These have created the need to discover new compounds endowed with antibacterial property, possibly, acting through mechanisms of action that are distinct from those of the well-known classes of antibacterial agents to which many clinically relevant pathogens are now resistant (Abhishek, Ravichandran, Madhvi, & Agrawal, 2010).
Gas chromatography's combination of speed, sensitivity, and high resolving power makes it an excellent technique for separating complex samples. Furthermore, it is simple to connect to spectrometry methods such as mass spectrometry (MS) to identify unknown compounds directly. Gas chromatography can quantitatively determine chemical compounds present at very low concentrations by using the flame ionization and electron capture detectors (both of which have very high sensitivities). It provides both universal and specific information on individual components in a sample and information on the identity of those components. The use of GC-MS is based on the realization that the numerous compounds in a plant can be grouped into only a few classes (alkaloids, flavonoids, phenolics, terpenoids and tannins of the classification system) and that each member of these classes can be characterized by a limited number of ions in the mass spectra. It proves to be a valuable method for the analysis of polar and non-polar compounds and volatile essential oil, fatty acids in the areas of food, beverage, flavor, and fragrance analysis, forensic and criminal cases, pesticides detection, in medical and pharmaceutical applications as well (Jayapriya & Gricilda, 2015; Sermakkani & Thangapandian, 2012).
Hence, thorough validations would provide alternative remedies for easily accessible and affordable to local individuals. It is against this background. The research wishes to determine the antimicrobial activity of Detarium microcarpum of stem bark and identify compounds that could be responsible for antimicrobial activity.
The genus Detarium (Fabaceae, subfamily Caesalpiniaceae) of the Detarieae tribe has initially been found in Africa, with three species, namely D. microcarpum Guill. (Perr), D. senegalense J. F. Gmelin and D. macrocarpum Harms (Peter, Chukwuemeka, Mbaoji, Nwabunike, & Onyeto, 2012). The ecological distribution of the species is different but morphologically looks similar (Akah, Nworu, Mbaoji, Nwabunike, & Onyeto, 2012). In various parts of Nigeria, the roots, bark, leaves and seeds of D. microcarpum are used to treat various diseases (Abreu & Relva, 2002; Burkill, 1995). Infusion or decoction of plant parts is used to treat urinary tract infections, haemorrhoids, malaria, leprosy, arthritis, digestive problems, pain relief, bronchitis and pneumonia (Abreu et al., 2002). A decoction of the leaves is used as a postpartum and hemostatic drug in some parts of the country and to treat seizures in some situations (Okwu & Uchegbu, 2009). In Kashere, the bark, leaves and roots are prepared to treat ailments such as skin infections, diarrhea and menstrual pain, rheumatism, urogenital infections and haemorrhoids. Considering the claims made by herbalists, the methanol crude extract of D. microcarpum was tested on isolates of bacteria associated with the claimed disease. This is to determine a single compound or group of plant compounds, using the GC-MS method to its advantage in identifying specific phytochemicals associated with current traditional claims and use as herbal medicines.
Materials and methods
Collection and Extraction
Two hundred and fifty grams of D. microcarpum stem bark were collected from Kashere and its environs, Gombe. The sample collected (voucher number FUKH 056) was identified by Professor A. M. Gani of the Department of Biological Sciences, Federal University of Kashere. The sample was air-dried at room temperature of 25 oC for ten days, and 150 g was blended into powder in a kitchen blender (Murugan et al., 2020). 100 g of the powder was flooded with 1 L of 95% ethanol for two weeks at room temperature. The filtrate after that was concentrated using a rotary evaporator (Yusha'u, Gabari, Dabon, Hassan, & Dahiru, 2014).
Test Organisms
E. coli,S. aureus, S. typhi and K. pneumonia, P. aeruginosa were obtained from the stock culture of the Department of Microbiology Laboratory, Bayero University Kano Nigeria and confirmed using cultural and biochemical characterization and maintained in a nutrient broth (NB) medium in a refrigerator. The inoculation density was standardized using a corresponding cell concentration of 1.0×108 CFU/mL equivalent to 5 % McFarland standard (Ramalivhana et al., 2014).
Antibacterial Assay
After the two-fold serial dilution method, broth dilution and disk diffusion techniques were used. Kirby-Bauer test (agar disk diffusion) was employed to give quantitative insight into the level of inhibition exhibited by the plant extract on each isolate. Six millimeters in diameter discs were primed using No. 1 Whatman Filter Paper. Each disc was sterilized in a hot air oven at 160 oC for 2 hrs after which it was impregnated with 0.2 mL of respective extracts (320, 160, 80, 40, 20, 10, 5, 2.5, 1.3, and 0.63 w/v) concentrations, using dimethyl sulfoxide (DMSO) as solvent (Delnavazi, Tavakoli, Rustaie, Batooli, & Yassa, 2014). MH agar plates seeded with test organisms were inoculated with impregnated discs of the ten varying concentrations and incubated at 37 °C for 18 h; then, one inhibition (ZI) was observed and recorded. Zones were compared with that of some standard antibiotics, as sensitive (S) intermediate (I) and resistant (R) as follows; Ampicillin 10 µg (S = ≥17, I = 14 – 16, R= ≤13) and Cotrimoxazole 1.25/23.75 µg (S= ≥16, I= 11-15, R= ≤10); for S. aureus, K. pneumonia, E. coli, S. typhi and Gentamicin 10 µg (S= ≥15, I= 13-14, R= ≤12) for P. aeruginosa following CLSI (2011) recommendation.
Minimum inhibitory concentration (MIC)
One millilitre of prepared concentrations of the extract was added to a 9 mL broth of MH after which1 mL of the test organism (standardized) was also added. Inoculated samples were then incubated at 37 °C for 18- 24 h, and observed visual increase in turbidity of the medium in the test tubes. The visual observation was made by comparing the inoculated and uninoculated tubes (Andrews, 2001).
Minimum Bactericidal/Bactriostatic Concentrations (MBC/MBS)
To determine MBC, the isolates in the medium, identified as MIC, were sub-cultured on fresh NA (nutrient agar) plates and incubated for 18-24 h at 37 OC for possible bacterial growth. A 99.9 % reduction in the colony count from the initial inoculum was recorded as minimum bactericidal concentration, while samples demonstrating growth were considered minimum bacteriostatic concentration (Saleem et al., 2015).
Preparation (GC-MS) and Identification of Components
The sample was extracted with methanol and analyzed using GC-MS (GC-MS-QP2010 Plus Shimadzu, Japan). The data were obtained on an Elite-1(100% Dimethylpolysiloxane) column (30 0.25 mm 1 μmdf). Helium (99.999 %) was used as the carrier gas with a 1 mL/min flow rate in the split mode (10:1). An aliquot of 2 μl of the sample's methanol solution was injected into the column at 250 oC through the injector. The linear velocity of 46.3 cm/sec, total 6.2mL/min and purge flow of 3.0 mL/min were set. GC oven temperature started at 80.0 ºC and held for 1 min and raised to 200 ºC at the rate of 10 ºC/min. With 4 min holding, the temperature was allowed up to 280 ºC withholding 5 min. Ion source temperature 230 ºC, interface 250 °C a solvent Cut Time of 2.50 min. The mass spectrum of compounds in samples was obtained by electron ionization at 70 eV, and the detector was operated in ACQ scan mode with a start of 40 mz and end at 600 mz. A scan start time of 3.00 s and end time of 28.00 min with a speed of 1250 was maintained (Devender & Ramakrishna, 2017).
Identification of components
The molecular structure, molecular mass and estimated fragment were used to identify the substances. The National Institute of Standards and Technology's database was used to interpret the mass spectrum GC-MS (NIST05s.LIB). Name, molecular weight, and structure of the phytocompounds determined with NIST library Version 2005.
RESULTS
Zones of inhibition
Antibacterial action of D. microcarpum stem bark was measured against S. aureus, P. aeruginosa, E. coli, S. typhi, and K. pneumonia and compared to standard Ampicillin 10 g/disc, Cotrimoxazole 1.25/23.75 g/disc, and Gentamicin 10 g/disc following CLSI (2011) recommendations. The concentrations measured ranged from 0.63 mg/Disc to 320 mg/Disc. The mean active concentration of the extract ranged between ≥ 106.7 ± 46.2 mg/disc to 293.3 ± 115.5 mg/disc among the bacteria tested. Furthermore, the actual ZID values were 20.0 ± 00 mm for E. coli, P. aeruginosa 20.0 ± 3.5 mm, and K. pneumoniae had 17.3 ± 6.1 mm. Both were statistically significant. The mean ZID of S. typhi was 17.3 ± 3.1 mm, and S. aureus 17.3 ± 2.1 mm showed a significant statistical difference(p< 0.05), as shown in Table 1.
There was a statistical difference in the active mean concentration between different bacteria. For example, the active mean concentration difference between E. coli and S. aureus was (p= 0.0002), K. pneumonia and S. aureus was (p = 0.0002), S. typhi and S. aureus were (p= 0.0014), P. aeruginosa and E. coli (p= 0.0014), P. aeruginosa and K. pneumonia was (p= 0.0014) and S. typhi and P. aeruginosa (p= 0.0109).
Table 1
Effect of D. microcarpum methanol crude extract on selected bacterial isolates.
MIC, MBC and MBS
The mean MIC of the methanol crude extract of D. microcarpum, which can inhibit the visible growth, was determined. The MIC among the tested bacteria ranged from 4.2 ± 1.4 mg/mL to 11.7 ± 7.6 mg/mL. There was no statistical significance difference in the mean MIC observed for S. aureus (11.7 ± 7.6 mg/mL), K. pneumonia (6.7 ± 2.9 mg/mL), P. aeruginosa (6.7 ± 2.9 mg/mL) and E. coli (5.0 ± 00 mg/mL) at p < 0.05, except for S. typhi (4.2 ± 1.4 mg/mL) whose mean MIC showed significance difference (p < 0.038) as presented in Table 2. The results of the MBC ranged between 5 ± 00 to 20 ± 00 mg/L and showed no statistically significant difference across the tested bacteria. MBS was only significant on S. aureus 16.7 ± 5.8 mg/L (p = 0.038), and the results ranged from 5 ± 00 to 16.7 ± 5.8 mg/L. Overall assessment of MBC/MIC ratio among the tested bacteria revealed less than ≤ 1:4 ratio effect of the D. microcarpum stem bark extract. A ratio of 1:1.82 for S. aureus, 1:2 for E. coli, 1: 2.98, K. pneumoniae and 1:0.75 for P. aeruginosa and 1:1.2 for S. typhi was observed as contained in the Table 2.
Table 2
Antibacterial activity of methanolic extract of D. microcarpum stem bark on some bacterial pathogens
Phytocompounds identification
During GC-MS analysis of the D. microcarpum stem bark extract, 26 phytochemical components (Figure 1) were discovered. The compounds were identified using the peak, area, retention time (RT), and molecular formula (WM), as indicated in Table 3. The first chemical found was (-)-L-Dibenzoyl-tartaric (RT = 6.37 min, MW = 358, RI = 2883), while the last compound detected was Lineoleoyl chloride (RT = 25.73 min, MW = 298, RI = 2130). At 20.21 RT, Oleic acid (C18H34O2) has a maximum volume of 40.37 percent, whereas 1, 3-Dimethyl-2,4-dinitrobenzene (C8H8N2O4) has a minimum volume of 0.2 percent. Others found include Oleoyl chloride (C18H33ClO) RT = 18.99 and 7.69 percent volume, 13-Octadecenal (Z) (C18H34O) RT = 23.57 and 5.6 percent volume, and Methyl 11-octadecenoate (C19H36O2) RT = 18.65 min, 4.42 percent volume, and 21 others in decreasing order.
Table 3
Phytocompounds identified from the methanol crude extract of D. microcarpum stem bark
Discussion
Detarium microcarpum species were reported to have shown considerable healing effect when the prepared decoction of either the stem barks or roots is administered as widely claimed by traditional healers (Mogana, Adhikari, Tzar, Ramliza, & Wiart, 2020). The stem bark extract showed potent (bactericidal) inhibition at varying concentrations in this work. The differences observed in the effect of the active concentration on respective bacteria could be relatively explained by the number and type of fatty acids (Knapp & Melly, 1986; Mcgaw, Jäger, & Staden, 2002) contained in the extract, as observed in Table 3.
Similarly, it was observed, long and medium chains of unsaturated fatty acids formed more than 50% of the fatty acid identified in this plant and were reported to be more active against gram-positive(S. aureus and P. aeruginosa) bacteria (Nguyen, Le, Phan, & Tran, 2017). Methyl esters of Arachidic acid, Dodecanoic acid 4-methyl- methyl, n-Hexadecanoic acid and stearic acid, Z-7-Tetradecenal, Glycerol 1,2-dipalmitate, Sucrose, Lineoleoyl chloride and Oleic acid are the most abundant compounds in this plant. Some investigators have also reported the abundance of these compounds in D. microcarpum and other plants and their bioactive properties. For example, (Ololade, Fakankun, Alao, & Ajewole, 2016) reported the significant inhibitory effects of essential oils extracted from Annona muricata (palmitic acid, oleic acid, cis- 9-hexadecanal, cis-9-octadecanal, pentadecanoic acid) against six multidrug-resistant bacteria, including S. typhimurium, P. aeruginosa, E. coli and S. aureus.
Oleic acid, Oleoyl chloride and palmitic acid and their derivatives here identified, and other fatty acids esters from the stem bark extract can modify bacterial cell membrane, thereby obstructing oxygen intake and transport of amino acids necessary for cell growth and repairs (Freese, Sheu, & Galliers, 1973). Oleic and Linoleic acids were reported to inhibit S. aureus but not active against K. pneumoniae, E. coli, and P. aeruginosa (Dilika, Bremner, & Meyer, 2000). Thus, the gram-positive bacteria tested bactericidal activity could be partly due to some of these fatty acids (Mustapha & Runner, 2016; Raval, Vaghela, Mandavia, & Andgolakiya, 2016) seen in Table 3. A synergistic action was also reported of activity of these identified compounds on the tested bacteria in bioactivity between oleic and linoleic acids and between linolenic acid and monoglyceride on inhibition of S. aureus (Dilika et al., 2000) as further evidence to support the potentials of long-chain fatty acid remained the possible factor for inhibition of bacteria (gram-positive) tested. However, susceptibility of gram's negative (E. coli, K. pneumonia and S. typhi) bacteria could not be explained in this context because they are affected by short-chain fatty (≤ 6 Carbon atoms) (Mcgaw et al., 2002)that were not observed. The susceptibility of bacteria (gram-negative) was influenced by pH and concentration of short-chain fatty acids of a plant ( ; McGaw et al., 2002a) (Freese et al., 1973; Mcgaw, Jäger, & Staden, 2002). Lineoleoyl chloride and 4-Dodecanol were earlier described as potent antimicrobial agents (Jenecius & Mohan, 2014; Saravanan, Chandramohan, Rani, & Deepa, 2013), while n-hexadecanoic acid has nematicide and pesticide effects (Jananie, Priya, & Vijayalakshmi, 2011).
Although high numbers of active bioactive compounds were identified, no individual compound was isolated and tested in this work and therefore could not conclude the specific reason for the activity. Nevertheless, to corroborate current findings with previous ones, many other researchers have previously reported the antibacterial activity of Detarium against common bacterial pathogens but did not identify the possible phyto-compounds involved (Abreu et al., 1998; Okwu et al., 2009; Sowemimo et al., 2011). Similarly, the susceptibility of these common pathogens toD.senegalense in addition to methanolic extract of the stem back of D. microcarpum species has also been reported byOkwu et al. (2009) and Olugbuyiro, Moody, and Hamann (2009).
Conclusion
This study has provided data on the chemical composition of D. microcapum, which is traditionally used to treat urogenital infections, haemorrhoids, intestinal tract infections and pains relievers, and the antibacterial activity of methanol crude extract of stem bark. To date, there are very few reports on the determination of chemical constituents from methanol crude extract of D. microcapum stem bark using chromatographic techniques. Here, the bioactivity and presence of some compounds GC-MS identified are reported. The antibacterial activity of the identified compounds directly correlates with the bioactivity of the same compound isolated elsewhere, giving a clear insight on the potential of the plant and supporting the claim for its traditional usage. This report provides for quality control of the herbal use of the plant-based on the concept of phyto-equivalence of herbs, validation of the natural herbal medicine and could be used for new drug formulation in combating the threat of drug resistance by pharmaceutical industries. However, the research recommends further and exhaustive research to ascertain actual active constituents as possible drug candidate.
